The tsetse fly is of high public health and agricultural significance due to its obligate vector role in the transmission of African trypanosomiasis. Trypanosomes (Trypanosoma brucei subspp.) rely on the tsetse fly for their development, recombination, multiplication and transmission. Tsetse flies also harbour two enteric bacterial symbionts, Wigglesworthia glossinidia and Sodalis glossinidius. It is believed that these microbial symbionts provide metabolic capabilities that enable tsetse's specialized lifestyle of exclusive blood feeding. Blood although rich in amino acids and lipids is deficient in many B vitamins, particularly thiamine. The overall objective of the current proposal is to examine symbiotic metabolic integration and its role in tsetse fly fitness and vector competence. The specific hypothesis behind the proposed research is that energy processing by Sodalis and Wigglesworthia serves as a critical factor in the microbial homeostasis of the tsetse fly with disruption altering host fitness and vector competence. This hypothesis is based on the following observations. First, the similar growth dynamics of Sodalis and Wigglesworthia during metabolically intensive periods of tsetse development supports overlapping roles in energy processing. Second, significant Wigglesworthia and Sodalis proliferation occurs at the teneral adult (i.e. newly eclosed) life stage, the developmental period of highest vector competence in wild type tsetse flies. Third, comparative genomic analyses as well as their physical proximity within the tsetse midgut, suggests that de novo thiamine (Vitamin B1) synthesis is exclusively performed by Wigglesworthia and subsequently acquired by Sodalis through a thiamine salvage pathway. Fourth, our preliminary data demonstrate that the proliferation of Sodalis density requires vitamins provided by an exogenous source. Fifth, our preliminary data also suggests that the Sodalis thiamine salvage pathway is inversely regulated by exogenous sources of thiamine, specifically thiamine monophosphate (TMP), both in culture and within the tsetse fly. To test our hypothesis, the functional regulation of the Sodalis thiamine salvage pathway will be examined through expression analyses and genetic manipulation. Tsetse flies, Glossina morsitans morsitans, will be reconstituted with Sodalis thiamine recruitment mutants and host fitness traits evaluated. Lastly, the effect of B-vitamin supplementation towards reducing the higher vector competence exhibited by non-teneral Wigglesworthia-free tsetse flies will be investigated. A greater understanding of the energy processing of tsetse fly symbionts expands our existing knowledge of tsetse fly biology and may stimulate alternative strategies towards combating African trypanosomiasis. PUBLIC HEALTH RELEVANCE: The tsetse fly is of high public health and agricultural significance due to its vector role in the transmission of African trypanosomiasis. Tsetse flies also harbour a limited digestive tract microbial community that are critical components of their host's biology and also co-localize with trypanosome infections. The proposed research expands our understanding of tsetse fly biology and offers potential novel avenues for vector control strategies.